The micromanipulation of microorganisms, including unicellular and multicellular microorganisms and cells permits the insertion of foreign materials into individual cells for genetic manipulation, cellular response quantification, or intracellular structure imaging. Possessing many advantages, mechanical cell injection is highly effective for delivering macromolecules and is free from concerns about phenotype alteration.
As cell injection is a labor intensive task, efforts for automating cell injection have been continuous. The vast majority of these systems were developed to facilitate the handling of mouse/Drosophila/zebrafish embryos/oocytes for genetics and reproduction applications (See Y. Sun and B. J. Nelson, “Biological cell injection using an autonomous microrobotic system,” Int. J. Robot. Res., Vol. 21, No. 10-11, pp. 861-868, 2002; L. Mattos, E. Grant, R. Thresher, and K. Kluckman, “New developments towards automated blastocyst microinjections,” in Proc. IEEE International Conference on Robotics and Automation (ICRA'2007), 2007; R. Kumar, A. Kapoor, and R. H. Taylor, “Preliminary experiments in robot/human cooperative microinjection,” Proc. IEEE International Conf on Intelligent Robots and Systems, pp. 3186-3191, Las Vegas, 2003; and H. Matsuoka, T. Komazaki, Y. Mukai, M. Shibusawa, H. Akane, A. Chaki, N. Uetake, and M. Saito, “High throughput easy microinjection with a single-cell manipulation supporting robot,” J. of Biotechnology, Vol. 116, pp. 185-194, 2005; W. H. Wang, X. Y. Liu, D. Gelinas, B. Ciruna, and Y. Sun, “A fully automated robotic system for microinjection of zebrafish embryos,” PLoS ONE, vol. 2, no. 9, p. e862, September 2007; and S. Zappe, M. Fish, M. P. Scott, and O. Solgaard, “Automated MEMS-based drosophila embryo injection system for high-throughput RNAi screens,” Lap Chip, Vol. 6, pp. 1012-1019, 2006).
In microrobotic injection of suspended cells (e.g., embryos/oocytes), cells must be immobilized, preferably into a regular pattern to minimize cell searching and switching tasks and increase injection speed. Differently, most mammalian cells (e.g., HeLa cells, fibroblasts, and endothelial cells) adhere to the bottom surface of a culture dish/plate during in vitro culture into an irregular pattern. Although adherent cells do not require immobilization efforts, they are highly irregular in morphology, which makes robust pattern recognition difficult and full automation challenging. Additionally, they are only a few micrometers thick, posing more stringent requirements in microrobotic positioning. The small thickness and large variations require precise determination of relative vertical positions between the micromanipulating device and the cell.
A microinjection system for microinjecting adherent cells is disclosed in Lukkari et al (Proc. 2005 IEEE International Symposium on Computational Intelligence in Robotics and Automation). The micromanipulator of the system, however, is a joystick-controlled semi-automatic device that necessitates an operator to control movement of an injecting device and the microinjection of the cells. Hence, the semi-automatic system of this disclosure is immune to operator proficiency variations and from human fatigue.
Currently, no automated, high-throughput adherent cell micromanipulation systems are known. Such automated systems can serve as an important tool in the biotech industry and will have significant implications in molecule testing and the creation of stem cell lines for individualized stem cell-based therapy.
In view of the foregoing, what is needed is a system and method for cellular micromanipulation that overcomes the limitations of the prior art, such that the system and method is capable of automation, provides robustness, high-throughput (including sample positioning), high success rates, and high reproducibility.